Direct PCR from E. coli colonies

John Nash nash at nrcbsa.bio.nrc.ca
Tue Jul 11 15:17:07 EST 1995


In article <DBJxqL.Azr at murdoch.acc.virginia.edu>,
William D. Woolfolk <wdw7m at virginia.edu> wrote:

>Yes, you can use a similar method.  I have frequently amplified directly 
>from bacteria by pulling up a small amount of bacterial material with a 
>pipet tip, and adding it to a 50ul PCR reaction with the Taq omitted.  I 
>heat to 95C for 15 minutes,then add Taq and cycle as I would for normal 
>DNA template-based PCR.  The only problem I ever had was using a massive 
>clump of bacteria, which decreased amplification some.  A small swipe is 
>more than adequate for PCR, so do not overdo it.  Good luck.

A 10 ul reaction works fine, and (in my hands) one doesn't need to
heat-treat for 15 min (but I do use a 2 min 95 deg first step after
adding Taq).  Just touching a micropipette tip to the colony (you
don't need to see the cells you've removed) and mixing it in with the
master mix also suffices to give nice bands.

john
-- 
John Nash                       Institute for Biological Sciences      *
Email: nash at nrcbsa.bio.nrc.ca   National Research Council of Canada  * .*
World Wide Web: http://cansnd.cisti.nrc.ca/~nash/home.html            *
         Disclaimer: Opinions are mine, not NRC's



More information about the Methods mailing list